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Abnormal cell proliferation in the epidermis of the fall webworm, Hyphantria cunea, induced by the infection of a nuclear-polyhedrosis virus
JOURNAL
OF INVERTEBRATE
Abnormal
PATHOLOGY
12,
310-320
(1968)
Cell Proliferation in the Epidermis of the Fall Webworm, Hyphantria cuneu, Induced by the Infection of a Nuclear-Polyhedrosis Virus HITOSHI WATANABE Facuhy
of Agrkdture,
University Tokyo, Japan
Bunkyo-ku, Received
February
8,
of Tokyo,
1NS
Histopathological studies of the larval integument of the fall webworm, Zlyphantria cuneu, infected with a nuclear-polyhedrosis virus were carried out. It was found that in the early stage of the disease most regions of the epidermis swelled to form multilayered structures as the result of the abnormal proliferation of the epidermal cells. During the multiplication period, different phases of amitotic figures were always found in the epidermis. After the amitosis, the resulting cells enlarged markedly with an initiation of polyhedral formation in their hypertrophied nuclei. Thus, the diseased epidermis in the middle stage of infection consisted of several layers of the cells packed with a large number of polyhedra. In the later stages of the disease, multilayered epidermis was completely disintegrated; the constituent cells were separated from each other and were released from the integument into the body cavity. About this time, darkly pigmented spots were occasionally observed on the body surface of moribund larvae. Histologically, these spots were cuticular projections containing muscle tissue, blood cells, fat-body tissue, and disintegrated epidermal cells, with necrotic and melanized scabs covering the outside.
In the course of an histopathological examination being conducted on various larval tissues of the fall webworm, Hyphantria cunea, infected with nuclear-polyhedrosis virus, the author observed that the diseased epidermis generally showed a multilayered structure resulting from abnormal cell proliferation. The purpose of the present paper is to present a description of the abnormal cell proliferation and to report the development and disintegration of the multilayered epidermis during the progress of the viral infection.
INTR~DU~~N Insect “tumors” associated with viral infections have been recognized so far in the larval midgut of the European spruce sawfly, Diprion hercyniae, afflicted with a nuclear polyhedrosis (Bird, 1949), and in the epidermis of the silkworm, Bombyx mori, infected with Tipula iridescent virus ( Hukuhara, 1964). Similarly, tumorlike structures in the adult midgut have been generally found in the cytoplasmic polyhedroses of Alsophila pometaria, Operophtern brumuta, and Paleacrita vernata (Neilson, 1965). It has been also reported that extensive proliferation of blood cells occurs in the Tipula paludosa larvae in the early stage of the nuclear polyhedrosis (Xeros, 1966). [See also the review of neoplasms in insects by Harshbarger and Taylor
MATERIALS
AND
METHODS
Larvae of the fall webworm, Hyphantria cunea, were obtained from field-collected eggs, and reared in the laboratory on mulberry leaves. The early fourth-instar larvae were allowed to feed for 24 hr on the leaves
(1968).] 310
ABSORXIAL
PROLIFERATIOS
contaminated with a suspension of nuclear polyhedra (5 X lo8 polyhedra/ml). The subsequent rearing was done at room temperature (2%28’C) by feeding on fresh leaves. To prepare the histological specimens, a series of diseased and control larvae were dissected at 24hr intervals, and body parts including epidermis, fat body, muscle, tracheal epithelium, etc., were fixed with Carnoy’s fluid. Sections were cut at 4 y, and stained with hematoxylin-eosin, Feulgenlight-green, and Giemsa after hydrolysis (10 min in 1.0 N HCl; at 60°C). RESULTS
AND
DISCUSSION
The integument of the normal fall-webworm larva consists, as in other lepidopterous larvae, of a single layer of epidermal cells and cuticle covering the epidermis. The nucleus of each epidermal cell is located in the basal region (Fig. 1). Gross observation of diseased larvae 1 or 2 days after the viral infection revealed that some regions of epidermis swelled several times larger than the normal (Fig. 2). The swelling of the epidermis was due to the formation of a multilayered structure resulting from an abnormal proliferation of the cells. Thus, during the earlier stages of viral infection, the epidermal cells inordinately increased in number accompanied by appreciable changes in the intracellular morphology as shown in Figs. 3, 4, 5, and 6. The diseased epidermal cell could be observed to multiply, going through the following phases: ( 1) enlargement of epiderma1 cell and hypertrophy of the nucleus; (2) granulation of nuclear chromatin increasing Feulgen-positive reaction; (3) concentration of nuclear chromatin in the center of the nucleus; (4) elongation and constriction of the nucleus as well as its containing chromatin cluster; (5) separation of the chromatin into two halves, one moving towards the apical periphery of the
OF
VIHUS-INFECTED
CELL
311
nucleus and the other moving to the basal direction; and (6) formation of cellular membrane between the two chromatin clusters to divide the cell body into two daughter cells. Throughout the multiplication period, separation of sister chromosomes in the nucleus of the proliferating cell could not be entirely observed, suggesting that the epidermal cells multiply rapidly and successively in an apparently amitotic system [as for the definition of amitosis, see Mazia (1961)l. Thus, in most regions of epidermis, the cells proliferated to form a multilayered structure growing into the body cavity. .ifter amitosis, the resulting cells enlarged markedly, following an initiation of polyhedral formation in the hypertrophied nuclei. Accordingly, the diseased epidermis in the middle stage of viral infection was composed of several layers of the cells packed with a large number of polyhedra (Figs. 710). From the foregoing results, it is evident that the abnormal proliferation of epiderma1 cells was induced by the viral infection and continued with the progress of the disease. Although the exact function of the virus in inducing the amitotic cell proliferation is still unknown, the inordinate reproduction of cells in the epidermis would appear to be one of the compensating reactions of the tissue for the internal damage by the irritant. It was similarly noted by Nagashima (unpublished) that in the externally wounded integument of Bombyx mori the epidermal cells around the affected area inordinately increased in number in an amitotic fashion. In the later stage of the disease, multilayered epidermis was completely disintegrated; the constituent cells were separated from each other and were eventually discharged from the integument into the body cavity (Figs. 11 and 12). However, the cuticle was not affected during the progress of the viral disease. Nevertheless, histologi-
312
WATANABE
FIG. 1. Healthy integument of the fourth-instar larva of the fall webworm, Hyphantria cunea. Hematoxylin-eosin stain. 470 X . FIG. 2. Diseased integument and fat body 1 day after the initiation of the viral infection. Note swelling of epidemk Hematoxylin-eosin stain. 120 X. FIG. 3. Diseased integument at an early stage of the viral infection. HCl-Giemsa stain. 470 X. FIGS. 4-6. Diseased integument at the second stage of the viral infection. Note different features of intranuclear change in the epidermal cells appeared during their multiplication. Hematoxylin-eosin stain. 470 X.
ABNORMAL
FIGS.
PROLIFERATION
7 and 8. Diseased integument and fat multilayered structure of epidermis consisted (Fig. 7) and HCl-Giemsa (Fig. 8) stains. The taining nuclei of epidermal cells. Polyhedra are 120 x. FIGS. 9 and 10. High-power magnification of The dark-staining shown in Fig. 8. HCl-Giemsa stain. FIGS. 11 and 12. Diseased integument in the loosening and sloughing of disintegrated epidermal 11) and HCl-Giemsa (Fig. 12) stains. 120 X .
Note
OF
VIRUS-IXFECTED
CELL
313
body in the middle stage of the viral infection. of polyhedra containing cells. Hematoxylin-eosin dark-staining areas in Fig. 8 indicate polyhedra conformed in almost all of the hypertrophied nuclei. diseased epidermis at the same infection stage as that bodies indicate polyhedra in the nuclei. 500 x . later stages of the viral infection. Note the general cells into the body cavity. Hematoxylin-cosin (Fig.
314
FIGS. 13 and 14. that these projections cells. Hematoxylin-eosin
WATANABE
Darkly pigmented cuticular projection in the integument of moribund are filled with muscle tissue, fat-body tissue, blood cells, and disintegrated (Fig, 13) and HCl-Giemsa (Fig. 14) stains. 120 x .
cal sections prepared from the moribund larvae occasionally showed the occurrence of darkly pigmented cuticular projections on the outer surface of the integument where the multilayered epidermis was completely disintegrated (Figs. 13 and 14). These spots contained muscle tissue, blood cells, fat-body tissue, and disintegrated epidermal cells, with necrotic and melanized scabs covering the outside. The formation
larva. Note epidermal
of the spots might be due to an internal body pressure, which pushes out a part of the defective integument without epidermis to form a cuticular projection there. The blood cells were accumulated in large masses around the affected area and melanization might occur as a reaction against the wound. Thus, although the virus-infected larvae of the fall webworm did show some specific
ABSOHMAL
PROLIFERATIOS
morphological characteristics in the epidermis, the histopathology of the other diseased tissues, such as the fat body and the tracheal epithelium, appeared to be typical of nuclear polyhedrosis of other lepidopterous insects. ACKNOWLEDGWENTS
The author wishes to express his thanks to Prof. H. Aruga, University of Tokyo, Dr. M. Kobayashi, Scricultural Experiment Station, Ministry of Agriculture and Forestry, and Dr. E. Nagashima, Shinshu University, all of whom generously offered helpful comments and suggestions for improving the manuscript. Appreciation also Miss hl. Hosaka for her assistance the histological sections.
is extended to in preparing
OF
VIRUS-ISFECTED
CELL
315
REFERENCES
1949. Tumor associated with a virus in an insect. Nature, 163, 777. HARSHBARGER, J. C., AND TAYLOR, R. L. 1968. Neoplasms of insects. Ann. Reu. Entomol., 13, 159-190. IIUKUHAHA, T. 1964. Induction of epidermal tumor in Bombyx mori (Linnaeus) with Tipula iridescent virus. J. In.~ct Puthol., 6, 246-248. MAZIA, D. 1961. Mitosis and the physiology of cell division. In “The Cell” (J. Brachet and A. E. Mirsky, eds.), Vol. 3, pp. 77412. Academic Press, Sew York. XEJLSON, M. M. 1965. Effects of a cytoplasmic polyhedrosis on adult Lepidoptera. J. Inuertebrute Puthol., 7, 306-314. XEHOS, N. 1966. The chromosomes and virogenic stroma in nucleopolyhedrosis of Tip& paludosu. J. Inuertebrate Pathol., 8, 240-249.
BIRD,
F. T. infection
OF INVERTEBRATE
Abnormal
PATHOLOGY
12,
310-320
(1968)
Cell Proliferation in the Epidermis of the Fall Webworm, Hyphantria cuneu, Induced by the Infection of a Nuclear-Polyhedrosis Virus HITOSHI WATANABE Facuhy
of Agrkdture,
University Tokyo, Japan
Bunkyo-ku, Received
February
8,
of Tokyo,
1NS
Histopathological studies of the larval integument of the fall webworm, Zlyphantria cuneu, infected with a nuclear-polyhedrosis virus were carried out. It was found that in the early stage of the disease most regions of the epidermis swelled to form multilayered structures as the result of the abnormal proliferation of the epidermal cells. During the multiplication period, different phases of amitotic figures were always found in the epidermis. After the amitosis, the resulting cells enlarged markedly with an initiation of polyhedral formation in their hypertrophied nuclei. Thus, the diseased epidermis in the middle stage of infection consisted of several layers of the cells packed with a large number of polyhedra. In the later stages of the disease, multilayered epidermis was completely disintegrated; the constituent cells were separated from each other and were released from the integument into the body cavity. About this time, darkly pigmented spots were occasionally observed on the body surface of moribund larvae. Histologically, these spots were cuticular projections containing muscle tissue, blood cells, fat-body tissue, and disintegrated epidermal cells, with necrotic and melanized scabs covering the outside.
In the course of an histopathological examination being conducted on various larval tissues of the fall webworm, Hyphantria cunea, infected with nuclear-polyhedrosis virus, the author observed that the diseased epidermis generally showed a multilayered structure resulting from abnormal cell proliferation. The purpose of the present paper is to present a description of the abnormal cell proliferation and to report the development and disintegration of the multilayered epidermis during the progress of the viral infection.
INTR~DU~~N Insect “tumors” associated with viral infections have been recognized so far in the larval midgut of the European spruce sawfly, Diprion hercyniae, afflicted with a nuclear polyhedrosis (Bird, 1949), and in the epidermis of the silkworm, Bombyx mori, infected with Tipula iridescent virus ( Hukuhara, 1964). Similarly, tumorlike structures in the adult midgut have been generally found in the cytoplasmic polyhedroses of Alsophila pometaria, Operophtern brumuta, and Paleacrita vernata (Neilson, 1965). It has been also reported that extensive proliferation of blood cells occurs in the Tipula paludosa larvae in the early stage of the nuclear polyhedrosis (Xeros, 1966). [See also the review of neoplasms in insects by Harshbarger and Taylor
MATERIALS
AND
METHODS
Larvae of the fall webworm, Hyphantria cunea, were obtained from field-collected eggs, and reared in the laboratory on mulberry leaves. The early fourth-instar larvae were allowed to feed for 24 hr on the leaves
(1968).] 310
ABSORXIAL
PROLIFERATIOS
contaminated with a suspension of nuclear polyhedra (5 X lo8 polyhedra/ml). The subsequent rearing was done at room temperature (2%28’C) by feeding on fresh leaves. To prepare the histological specimens, a series of diseased and control larvae were dissected at 24hr intervals, and body parts including epidermis, fat body, muscle, tracheal epithelium, etc., were fixed with Carnoy’s fluid. Sections were cut at 4 y, and stained with hematoxylin-eosin, Feulgenlight-green, and Giemsa after hydrolysis (10 min in 1.0 N HCl; at 60°C). RESULTS
AND
DISCUSSION
The integument of the normal fall-webworm larva consists, as in other lepidopterous larvae, of a single layer of epidermal cells and cuticle covering the epidermis. The nucleus of each epidermal cell is located in the basal region (Fig. 1). Gross observation of diseased larvae 1 or 2 days after the viral infection revealed that some regions of epidermis swelled several times larger than the normal (Fig. 2). The swelling of the epidermis was due to the formation of a multilayered structure resulting from an abnormal proliferation of the cells. Thus, during the earlier stages of viral infection, the epidermal cells inordinately increased in number accompanied by appreciable changes in the intracellular morphology as shown in Figs. 3, 4, 5, and 6. The diseased epidermal cell could be observed to multiply, going through the following phases: ( 1) enlargement of epiderma1 cell and hypertrophy of the nucleus; (2) granulation of nuclear chromatin increasing Feulgen-positive reaction; (3) concentration of nuclear chromatin in the center of the nucleus; (4) elongation and constriction of the nucleus as well as its containing chromatin cluster; (5) separation of the chromatin into two halves, one moving towards the apical periphery of the
OF
VIHUS-INFECTED
CELL
311
nucleus and the other moving to the basal direction; and (6) formation of cellular membrane between the two chromatin clusters to divide the cell body into two daughter cells. Throughout the multiplication period, separation of sister chromosomes in the nucleus of the proliferating cell could not be entirely observed, suggesting that the epidermal cells multiply rapidly and successively in an apparently amitotic system [as for the definition of amitosis, see Mazia (1961)l. Thus, in most regions of epidermis, the cells proliferated to form a multilayered structure growing into the body cavity. .ifter amitosis, the resulting cells enlarged markedly, following an initiation of polyhedral formation in the hypertrophied nuclei. Accordingly, the diseased epidermis in the middle stage of viral infection was composed of several layers of the cells packed with a large number of polyhedra (Figs. 710). From the foregoing results, it is evident that the abnormal proliferation of epiderma1 cells was induced by the viral infection and continued with the progress of the disease. Although the exact function of the virus in inducing the amitotic cell proliferation is still unknown, the inordinate reproduction of cells in the epidermis would appear to be one of the compensating reactions of the tissue for the internal damage by the irritant. It was similarly noted by Nagashima (unpublished) that in the externally wounded integument of Bombyx mori the epidermal cells around the affected area inordinately increased in number in an amitotic fashion. In the later stage of the disease, multilayered epidermis was completely disintegrated; the constituent cells were separated from each other and were eventually discharged from the integument into the body cavity (Figs. 11 and 12). However, the cuticle was not affected during the progress of the viral disease. Nevertheless, histologi-
312
WATANABE
FIG. 1. Healthy integument of the fourth-instar larva of the fall webworm, Hyphantria cunea. Hematoxylin-eosin stain. 470 X . FIG. 2. Diseased integument and fat body 1 day after the initiation of the viral infection. Note swelling of epidemk Hematoxylin-eosin stain. 120 X. FIG. 3. Diseased integument at an early stage of the viral infection. HCl-Giemsa stain. 470 X. FIGS. 4-6. Diseased integument at the second stage of the viral infection. Note different features of intranuclear change in the epidermal cells appeared during their multiplication. Hematoxylin-eosin stain. 470 X.
ABNORMAL
FIGS.
PROLIFERATION
7 and 8. Diseased integument and fat multilayered structure of epidermis consisted (Fig. 7) and HCl-Giemsa (Fig. 8) stains. The taining nuclei of epidermal cells. Polyhedra are 120 x. FIGS. 9 and 10. High-power magnification of The dark-staining shown in Fig. 8. HCl-Giemsa stain. FIGS. 11 and 12. Diseased integument in the loosening and sloughing of disintegrated epidermal 11) and HCl-Giemsa (Fig. 12) stains. 120 X .
Note
OF
VIRUS-IXFECTED
CELL
313
body in the middle stage of the viral infection. of polyhedra containing cells. Hematoxylin-eosin dark-staining areas in Fig. 8 indicate polyhedra conformed in almost all of the hypertrophied nuclei. diseased epidermis at the same infection stage as that bodies indicate polyhedra in the nuclei. 500 x . later stages of the viral infection. Note the general cells into the body cavity. Hematoxylin-cosin (Fig.
314
FIGS. 13 and 14. that these projections cells. Hematoxylin-eosin
WATANABE
Darkly pigmented cuticular projection in the integument of moribund are filled with muscle tissue, fat-body tissue, blood cells, and disintegrated (Fig, 13) and HCl-Giemsa (Fig. 14) stains. 120 x .
cal sections prepared from the moribund larvae occasionally showed the occurrence of darkly pigmented cuticular projections on the outer surface of the integument where the multilayered epidermis was completely disintegrated (Figs. 13 and 14). These spots contained muscle tissue, blood cells, fat-body tissue, and disintegrated epidermal cells, with necrotic and melanized scabs covering the outside. The formation
larva. Note epidermal
of the spots might be due to an internal body pressure, which pushes out a part of the defective integument without epidermis to form a cuticular projection there. The blood cells were accumulated in large masses around the affected area and melanization might occur as a reaction against the wound. Thus, although the virus-infected larvae of the fall webworm did show some specific
ABSOHMAL
PROLIFERATIOS
morphological characteristics in the epidermis, the histopathology of the other diseased tissues, such as the fat body and the tracheal epithelium, appeared to be typical of nuclear polyhedrosis of other lepidopterous insects. ACKNOWLEDGWENTS
The author wishes to express his thanks to Prof. H. Aruga, University of Tokyo, Dr. M. Kobayashi, Scricultural Experiment Station, Ministry of Agriculture and Forestry, and Dr. E. Nagashima, Shinshu University, all of whom generously offered helpful comments and suggestions for improving the manuscript. Appreciation also Miss hl. Hosaka for her assistance the histological sections.
is extended to in preparing
OF
VIRUS-ISFECTED
CELL
315
REFERENCES
1949. Tumor associated with a virus in an insect. Nature, 163, 777. HARSHBARGER, J. C., AND TAYLOR, R. L. 1968. Neoplasms of insects. Ann. Reu. Entomol., 13, 159-190. IIUKUHAHA, T. 1964. Induction of epidermal tumor in Bombyx mori (Linnaeus) with Tipula iridescent virus. J. In.~ct Puthol., 6, 246-248. MAZIA, D. 1961. Mitosis and the physiology of cell division. In “The Cell” (J. Brachet and A. E. Mirsky, eds.), Vol. 3, pp. 77412. Academic Press, Sew York. XEJLSON, M. M. 1965. Effects of a cytoplasmic polyhedrosis on adult Lepidoptera. J. Inuertebrute Puthol., 7, 306-314. XEHOS, N. 1966. The chromosomes and virogenic stroma in nucleopolyhedrosis of Tip& paludosu. J. Inuertebrate Pathol., 8, 240-249.
BIRD,
F. T. infection